Hi my name is Samantha. I am going to be a senior at San Ramon Valley High School this upcoming school year. As of right now, I am leaning towards majoring in Biology when I go to college with major interest in genetics and diseases. I have taken Honors Chem, regular bio, and AP Biology and plan on taking Honors Physics next year. In AP Bio I did a research project on CRISPR, how it works, and the ways CRISPR can be used to treat diseases and create designer babies. I am also a Varsity diver on my high school team, participate in Youth and Government, and coach gymnastics at Edge Gymnastics.
Using CRISPR and Gene Therapy to Help Prevent HIV By: Samantha Foon
Worldwide, people become infected and suffer from HIV. In 2015, around 1.1 million people died from an HIV related disease and around 37 million people were living with the HIV disease. HIV ranges from having flu-like symptoms like fever and having a sore throat, however, because HIV eventually leads to AIDS, symptoms like weight loss, recurrent infection, fatigue, and even death can be experienced.
HIV stands for Human Immunodeficiency Virus and will eventually lead to Acquired Immunodeficiency Virus or AIDS. HIV is a devastating illness because the virus attacks CD4 cells, or white blood cells, in the human body. Playing a major role in the immune system, CD4 cells are responsible for fighting off other diseases and viruses. Because HIV is a retrovirus, it uses the CD4 cells to replicate and then destroy the cell after replication.
CRISPR CRISPR stands for Clustered Regularly Interspaced Short Palindromic Repeat. CRISPR technology is used to make precise edits to DNA which allows scientists to remove specific genes without harming the genes around them. CRISPR uses a specific RNA sequence to target the Cas9 protein and guide it to the location of the gene that needs to be modified, in which the Cas9 and RNA together create a double strand break in the DNA that triggers the inside DNA repair system, leading to the genome to be modified.
Cas9 is CRISPR associated protein 9 and it is a RNA guided DNA Endonuclease. An endonuclease is an enzyme that cleaves the phosphodiester bond within the polynucleotide chain. CRISPR also functions as the immune system of simple life forms such as bacteria and microorganisms. CRISPR immune system attacks the genome of the invading virus. First viral DNA is inserted into the CRISPR sequence as spacers, then CRISPR RNA is produced, and then the CRISPR RNA will guide molecular machinery to attack the invading viral genome.
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Gene Therapy and Retroviral Vectors Gene therapy is a technique that scientists use involving the use of genes to treat or prevent diseases. In order to accomplish this, scientists can replace a mutated gene with a healthy copy of that same gene, “knock out” or inactivate the mutated gene, or introduce a new gene to the genome to help fight the disease. Because directly inserting a gene into a cell will most likely result in that gene not functioning, scientists use vectors to transfer the genes.
Retroviral vectors originally start off as a retrovirus. Scientists can create a retroviral vector by removing the gag, pol, and env genes. The gag gene, or group-specific antigen gene, codes for core structural proteins of the virus. The pol gene, or polymerase, codes for the enzymes reverse transcriptase, protease, and integrase. The env gene, or envelope gene, codes for the retroviral coat proteins. Once these genes are removed they are replaced with a therapeutic gene.
In order to produce the retroviral vectors, scientists need to use a packaging cell. A packaging cell line provides all of the viral proteins that are required for capsid production and the maturation of the vector. These packaging cell lines contain the gag, pol, and env genes.
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Using a Retroviral Vector and CRISPR to Genetically Engineer HIV Resistance in CD4 Cells
CRISPR will target the CCR5 and CXCR4 genes. These genes code for a protein on the surface of white blood cells (CD4 cells) and which acts as a receptor for chemokines. Chemokines are signaling proteins that are secreted by the cell. HIV mainly uses the CCR5 protein to enter host cells.
By using CRISPR to target the CCR5 and CXCR4 genes, host cells will no longer possess the receptor proteins that HIV uses to attach to host cells. Once these HIV co-receptors are removed, primary cells will have been edited with HIV resistance.
Using a retroviral vector to transfer CRISPR to white blood cells in the bloodstream will engineer the white blood cells with HIV resistance. To test this technique, scientists will use cultured CD4 cells in a lab in order to see if the retroviral vector successfully transferred CRISPR to the white blood cells and if CRISPR successfully edited out the CCR5 and CXCR4 genes.
In order for this design to work, I would have to find a way for the retroviral vector to locate the white blood cells and not the red blood cells in the bloodstream. I would also need to find a retrovirus that would be able to attach and transfer CRISPR into white blood cells that do not possess the CCR5 protein. This retrovirus would also have to be compatible with the CRISPR genome.
Potential Problems One potential problem with this design is that CRISPR/Cas9 could accidentally target a different gene with the same nucleotide sequence as either the CCR5 or CXCR4 gene. There is also the potential that the retroviral vector could target a different type of cell other than a white blood cell, resulting in the transfer of CRISPR into a completely different cell. Removing a receptor protein from the surface of a white blood cell could also effect cell signaling.
Hi my name is Samantha. I am going to be a senior at San Ramon Valley High School this upcoming school year. As of right now, I am leaning towards majoring in Biology when I go to college with major interest in genetics and diseases. I have taken Honors Chem, regular bio, and AP Biology and plan on taking Honors Physics next year. In AP Bio I did a research project on CRISPR, how it works, and the ways CRISPR can be used to treat diseases and create designer babies. I am also a Varsity diver on my high school team, participate in Youth and Government, and coach gymnastics at Edge Gymnastics.
Using CRISPR and Gene Therapy to Help Prevent HIV
By: Samantha Foon
Worldwide, people become infected and suffer from HIV. In 2015, around 1.1 million people died from an HIV related disease and around 37 million people were living with the HIV disease. HIV ranges from having flu-like symptoms like fever and having a sore throat, however, because HIV eventually leads to AIDS, symptoms like weight loss, recurrent infection, fatigue, and even death can be experienced.
HIV stands for Human Immunodeficiency Virus and will eventually lead to Acquired Immunodeficiency Virus or AIDS. HIV is a devastating illness because the virus attacks CD4 cells, or white blood cells, in the human body. Playing a major role in the immune system, CD4 cells are responsible for fighting off other diseases and viruses. Because HIV is a retrovirus, it uses the CD4 cells to replicate and then destroy the cell after replication.
CRISPR
CRISPR stands for Clustered Regularly Interspaced Short Palindromic Repeat. CRISPR technology is used to make precise edits to DNA which allows scientists to remove specific genes without harming the genes around them. CRISPR uses a specific RNA sequence to target the Cas9 protein and guide it to the location of the gene that needs to be modified, in which the Cas9 and RNA together create a double strand break in the DNA that triggers the inside DNA repair system, leading to the genome to be modified.
Cas9 is CRISPR associated protein 9 and it is a RNA guided DNA Endonuclease. An endonuclease is an enzyme that cleaves the phosphodiester bond within the polynucleotide chain.
CRISPR also functions as the immune system of simple life forms such as bacteria and microorganisms. CRISPR immune system attacks the genome of the invading virus. First viral DNA is inserted into the CRISPR sequence as spacers, then CRISPR RNA is produced, and then the CRISPR RNA will guide molecular machinery to attack the invading viral genome.
Gene Therapy and Retroviral Vectors
Gene therapy is a technique that scientists use involving the use of genes to treat or prevent diseases. In order to accomplish this, scientists can replace a mutated gene with a healthy copy of that same gene, “knock out” or inactivate the mutated gene, or introduce a new gene to the genome to help fight the disease. Because directly inserting a gene into a cell will most likely result in that gene not functioning, scientists use vectors to transfer the genes.
Retroviral vectors originally start off as a retrovirus. Scientists can create a retroviral vector by removing the gag, pol, and env genes. The gag gene, or group-specific antigen gene, codes for core structural proteins of the virus. The pol gene, or polymerase, codes for the enzymes reverse transcriptase, protease, and integrase. The env gene, or envelope gene, codes for the retroviral coat proteins. Once these genes are removed they are replaced with a therapeutic gene.
In order to produce the retroviral vectors, scientists need to use a packaging cell. A packaging cell line provides all of the viral proteins that are required for capsid production and the maturation of the vector. These packaging cell lines contain the gag, pol, and env genes.
Using a Retroviral Vector and CRISPR to Genetically Engineer HIV Resistance in CD4 Cells
CRISPR will target the CCR5 and CXCR4 genes. These genes code for a protein on the surface of white blood cells (CD4 cells) and which acts as a receptor for chemokines. Chemokines are signaling proteins that are secreted by the cell. HIV mainly uses the CCR5 protein to enter host cells.
By using CRISPR to target the CCR5 and CXCR4 genes, host cells will no longer possess the receptor proteins that HIV uses to attach to host cells. Once these HIV co-receptors are removed, primary cells will have been edited with HIV resistance.
Using a retroviral vector to transfer CRISPR to white blood cells in the bloodstream will engineer the white blood cells with HIV resistance. To test this technique, scientists will use cultured CD4 cells in a lab in order to see if the retroviral vector successfully transferred CRISPR to the white blood cells and if CRISPR successfully edited out the CCR5 and CXCR4 genes.
In order for this design to work, I would have to find a way for the retroviral vector to locate the white blood cells and not the red blood cells in the bloodstream. I would also need to find a retrovirus that would be able to attach and transfer CRISPR into white blood cells that do not possess the CCR5 protein. This retrovirus would also have to be compatible with the CRISPR genome.
Potential Problems
One potential problem with this design is that CRISPR/Cas9 could accidentally target a different gene with the same nucleotide sequence as either the CCR5 or CXCR4 gene. There is also the potential that the retroviral vector could target a different type of cell other than a white blood cell, resulting in the transfer of CRISPR into a completely different cell. Removing a receptor protein from the surface of a white blood cell could also effect cell signaling.
Sources
https://www.avert.org/about-hiv-aids/what-hiv-aids
http://www.who.int/features/qa/71/en/
http://www.genecards.org/cgi-bin/carddisp.pl?gene=CCR5
http://www.aidsmap.com/Gene-therapy-snips-HIV-out-of-infected-cells-and-makes-uninfected-cells-resistant/page/3046950/
http://www.news-medical.net/health/Gene-Therapy-Vectors.aspx
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3182074/
http://viracore.ucsf.edu/tidbits-of-lenti
https://ghr.nlm.nih.gov/primer/therapy/genetherapy
http://learn.genetics.utah.edu/content/stemcells/sctoday/
http://www.genetherapynet.com/viral-vector/retroviruses.html